High pressure, a tool for exploring heme protein active sites

Area of Science:

• Biochemistry
• Biophysics
• Protein Science

Background:

• High pressure is a valuable tool for studying protein dynamics and stability.
• Cytochrome P450s are a large family of heme proteins suitable for high-pressure investigations.
• Understanding pressure effects provides insights into volumic (ΔV°) and energetic (ΔG°) parameters.

Purpose of the Study:

• To investigate pressure-induced spin transitions and inactivation in cytochrome P450s.
• To compare reaction volumes of these processes across various analog-bound P450s.
• To correlate reaction volumes with protein properties like heme pocket hydration and substrate binding.

Main Methods:

• High pressure techniques were employed to study protein behavior.
• Characterization of pressure-induced spin transitions and P450 to P420 inactivation.
• Analysis of reaction volumes and their dependence on substrate analogs.

Main Results:

• Spin and inactivation volumes are influenced by substrate analogs, affecting heme pocket polarity and hydration.
• Linear correlations were observed between reaction volumes and protein properties (tyrosine exposure, hydration, substrate mobility).
• Monomeric P450s exhibit different pressure behaviors compared to oligomeric and heterooligomeric forms.

Conclusions:

• Substrate analogs significantly modulate the thermodynamic parameters of pressure-induced events in P450s.
• Heme pocket hydration and substrate interactions are major contributors to reaction volumes.
• Oligomeric structure and subunit interactions critically influence P450 stability, activity, and reductase recognition.